Device for the determination of glycated hemoglobin

ABSTRACT

An electrochemical device for determining the percentage of glycated hemoglobin in a blood sample is provided. The device includes a cathode and anode and one or more cells. The device may include an enzyme capable of reducing oxygen to water for determining the total amount of hemoglobin in a sample by electrochemically measuring, in an oxygen electroreduction reaction at a cathode, the amount of oxygen in the sample. The device may also be used to determine the amount of glycated hemoglobin in the sample (e.g., spectrometrically or electrochemically). Also provided are devices that include glycated hemoglobin hydrolysis agents or glycated hemoglobin separating agents.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.10/146,577, entitled “Method for the Determination of GlycatedHemoglobin”, filed May 14, 2002, which claims priority from U.S.provisional patent application No. 60/291,361, entitled “Biofuel Cell,”filed May 16, 2001, as well as U.S. provisional application No.60/377,886, entitled “Miniature Biological Fuel Cell That is OperationalUnder Physiological Conditions”, filed May 2, 2002, naming inventorsHeller, Mano, Kim, Zhang and Mao, the contents of which applications areincorporated herein by reference in their entireties.

BACKGROUND

1. Field of the Invention

The present invention relates to a process for the determination of theamount of irreversibly glycated hemoglobin, or HbA1c, present in asample of blood, relative to the amount of total hemoglobin. Inparticular, the invention incorporates in the method an electrochemical,enzyme-catalyzed reaction or reactions. The present invention alsorelates to devices associated with such processes or methods.

2. Background Information

HbA1c is a glycated hemoglobin formed by a binding reaction between anamine group of hemoglobin and the glucose aldehyde group, for examplebetween the amino group of the N-terminal valine of the β-chain ofhemoglobin and the glucose aldehyde group. The binding reaction firstforms a Schiff's base and then a stable ketoamine by Amadorirearrangement. The percentage of HbA1c (i.e. the amount of glycatedhemoglobin relative to total hemaglobin in the blood) has come to betaken as a measure of the level of blood glucose control a diabeticpatient has maintained for a period of two or three months prior to themeasurement. As such, percentage HbA1c has become an importantmeasurement by which health care providers can assist diabetic patientsin their care.

There are many known assays that can be used to determine HbA1cpercentage. In recent years research efforts have focused on creatingassays that are both highly accurate and fast. However, known HbA1cassays typically require a substantial number of time-consuming stepswherein the blood components must be separated and treated.

In the health care context, a diabetic patient is typically guided by aphysician to obtain an HbA1c measurement when the physician realizesthat there is a need for such information during an office visit. Thepatient then provides a blood sample to a laboratory and results arereturned to the physician hours or days later. Typically, the lab willuse a table top analyzer of the type presently available commercially.This time lag between the patient's visit and the result of the testrequires that the physician review the result long after the patient hasleft the office. If the physician believes that further consultationwith the patient is required in light of the test result, the patientmust be contacted again.

Currently, there is a device sold under the name “A1c NOW” by Metrika,Inc. of Sunnyvale, Calif. This handheld and disposable device (based ontechnology described in U.S. Pat. No. 5,837,546 entitled “ElectronicAssay Device and Method,” incorporated herein by reference) is said toprovide an HbA1c test result in eight minutes using a relatively smallsample of blood. The A1c NOW device is an example of the market demandfor a fast method of providing an HbA1c result for either home ordoctor's office use. However, the A1c NOW device is not as accurate assome laboratory assays. Thus, research has continued to focus on findinga highly accurate HbA1c assay that is also fast enough and simple enoughto permit a diabetic and his or her doctor to take a blood sample duringan office visit and have a trustworthy HbA1c measurement available fordiscussion in the same visit.

SUMMARY OF THE INVENTION

The present invention comprises a method of determining the amount orpercentage of glycated hemoglobin in blood or a sample derived fromblood, in which at least one of the assay steps is performedelectrochemically. The use of electrochemical methodology can retain orimprove the accuracy of other methods and potentially speed the ultimatedetermination. Devices providing electrochemical measurements can alsobe relatively small.

In one embodiment, the method includes electrochemically determining thetotal amount of hemoglobin in a sample by electrochemically measuring,in an oxygen electroreduction reaction at a cathode, the amount ofoxygen in the sample, preferably after it was exposed to air so as toassure that the hemoglobin is oxygenated. Because the amount of oxygendissolved in aerated physiological buffer at the assay temperature inthe absence of hemoglobin, termed here free oxygen, is known, the totalhemoglobin may be determined by subtracting the amount of free oxygenfrom the total oxygen measured, recognizing the fast equilibrium Hb+O₂

HbO₂. Electrochemically determining the total hemoglobin value can befollowed by a determination of the amount of glycated hemoglobin in thesample. In the process of the invention, the cathode reaction isaccomplished by contacting the sample with an enzyme. In thisembodiment, the enzyme can be a copper-containing enzyme, containingfour copper ions per active unit. The family of these enzymes includes,for example, laccases and bilirubin oxidases.

The glycated hemoglobin can be determined in different ways. In oneembodiment, the glycated hemoglobin is separated from the sample, forexample by capturing it with immobilized antibodies against HbA1c orwith a boronic acid modified surface. Examples of surfaces include thoseof small magnetic, polymer or glass beads. The percentage HbA1c can thenbe determined by either measuring the hemoglobin left in the sample fromwhich the HbA1c has been removed, or by measuring the amount of glycatedhemoglobin in the separated portion of the sample. The amount ofglycated hemoglobin can be measured spectrophotometrically, or by anelectrochemical measurement in the same manner as the total hemoglobin.In another embodiment the hemoglobin is hydrolyzed by an establishedmethod, such as digestion with a proteolytic enzyme. The ketoamines inthe hydrolyzate, such as the fragments comprising the Amadorirearrangement products of the Schiff base formed of amino acids,including valine and glucose, are then determined, preferably by anelectrochemical method. In the electrochemical method, theelectrooxidation of the hydrolyzed Amadori rearrangement product may becatalyzed by an enzyme and a dissolved or immobilized redox mediator.The enzyme can be, for example, a fructosamine oxidase, a fourcopper-ion containing copper enzyme such as a laccase or a bilirubinoxidase, ceruloplasmin, or ascorbate oxidase. The redox mediators canbe, for example, complexes of Os^(2+/3+), or of Ru^(2+/3+).

The present invention also comprises a device associated with processesor methods disclosed herein.

DETAILED DESCRIPTION

The invention incorporates one or more electrochemical steps in themethod of determining percentage HbA1c. The method of the invention isbased on the understanding that hemoglobin, being the oxygen carrier ofblood, reversibly binds oxygen, forming HbO₂. The equilibrium Hb+O₂

HbO₂ is rapid. Because O₂ is rapidly released by HbO₂ when O₂ isdepleted from the solution in an electrochemical cell, it is possible todetermine the concentration of HbO₂ in light of the reaction4H⁺+4e⁻+HbO₂→2H₂O+Hb.

Determining Total Hemoglobin Electrochemically

In the invention, it may be useful to pre-treat a blood sample bycollecting the relatively large blood cells on a filtration membrane.After rinsing the collected cells with saline to remove adheringproteins, the cell membranes may be ruptured by exposing them tode-ionized water or a detergent. In this manner, the dissolvedhemoglobin will pass the filtration membrane. The cell membranes willremain on the filter paper.

In a preferred form of the invention, total hemoglobin is thendetermined from the sample by electroreducing the oxygen bound to thehemoglobin to water at the cathode in an electrochemical cell. Theoxygen electroreduction catalyst preferably comprises a so-called“copper” enzyme such as bilirubin oxidase, a laccase, or an ascorbateoxidase.

The catalyst may further comprise a redox mediator to form a “wiredenzyme” arrangement. In this system, the electrical connection isbetween a cathode of the electrochemical cell and the oxygen reductioncatalyzing enzyme, especially a copper-containing enzyme, such asbilirubin oxidase (sometimes referenced herein as BOD). Thus, in oneform of the invention, it is preferred to “wire” reaction centers of anenzyme, e.g. bilirubin oxidase, to a cathode. Bilirubin oxidasecatalyzes the four-electron reduction of oxygen to water. A cathodeconstructed with bilirubin oxidase is especially preferred as the redoxenzyme can function under relatively neutral pH conditions. However,other enzymes (e.g. laccase) may be useful in the method of theinvention so long as they provide catalytic functionality for thereduction of oxygen to water.

Thus, the concentration of HbO₂ can be measured by the reaction4H⁺+4e⁻+HbO₂→2H₂O+Hb. This measurement may be done coulometrically. Theconcentration of available oxygen in arterial blood tends to be about 8mM. Because the concentration of O₂ in water in equilibrium with air at25° C. is known (the concentration is generally around 0.24 mM), theamount of non-Hb bound O₂ can then be subtracted in calculating theamount of HbO₂.

A cathode useful in the invention effectuates the four-electronelectroreduction of O₂ to water. The blue, copper-containing oxidases,examples of which include laccases, ascorbate oxidase, ceruloplasmine,and bilirubin oxidase, catalyze the four-electron reduction of O₂ towater. The preferred enzymes are exemplified by bilirubin oxidases,which unlike laccases, retain their more than 80%, and usually retainmore than 90%, of the maximal activity under physiological pH. Thecatalytic reduction of O₂ to water depends on the coordination of thefour Cu^(+/2+) ions of the enzymes. The Cu^(+/2+) ions are classified,by their ligands, into three “types”, types 1, 2, and 3. Type 1Cu^(+/2+) centers show an intense Cys S to Cu(2) charge transfer band ataround 600 nm; the site accepts electrons from an organic substrate,such as a phenol, ascorbate, or bilirubin, and relays the electrons tothe O₂-reduction site. The O₂-reduction site is a tri-nuclear cluster,consisting of one type 2 Cu^(+/2+) center and a pair of type 3 Cu^(+/2+)centers, their spectrum showing a shoulder at 330 nm.

There are different forms of bilirubin oxidase available, such asbilirubin oxidase from Myrothecium verrucaria (Mv-BOD) and bilirubinoxidase from Trachyderma tsunodae (Tt-BOD). Bilirubin oxidases areusually monomeric proteins and have molecular weights approximatelyranging from about 52 kDa to about 65 kDa. Tt-BOD is a monomeric proteinwith a molecular weight of approximately 64 kDa, while that of Mv-BOD isabout 52 kDa. Both Mv-BOD and Tt-BOD are multicopper oxidases, eachcontaining one type 1, one type 2, and two type 3 copper ions. Thesethree types are defined by their optical and magnetic properties. Type 1(blue) copper ions have a characteristic Cys to Cu (2) charge-transferband near 600 nm. The type 1 copper center accepts electrons from theelectron-donating substrate of the enzyme and relays these to the O₂reduction site. The latter is a trinuclear cluster, consisting of a type2 copper ion and a type 3 pair of cupric ions with a characteristic 330nm shoulder in its absorption spectrum.

In one embodiment of the invention, bilirubin oxidase from Myrotheciumverrucaria could be used in a cathode electrocatalyst layer. In acathode constructed using Mv-BOD, the electrostatic adduct of thepoly-anionic Mv-BOD and its “wire”, the polycationic redox copolymer ofpolyacrylamide (PAA) and poly(N-vinylimidazole) (PVI) complexed with [Os(4,4″-dichloro-2,2′-bipyridine)₂Cl]^(+/2+), are immobilized on thecathode.

In another embodiment of the invention, bilirubin oxidase (BOD) fromTrachyderma tsunodae can be used in a cathode electrocatalyst layer. InTt-BOD all of the ligands of the Type 2 and Type 3 Cu^(+/2+) centers areHis (histidines), similar to ascorbate oxidase. It is believed that thefull histidine coordination of the type 2 Cu^(+/2+) center is theunderlying cause of the relative insensitivity of bilirubin oxidases toinhibition by the chloride and hydroxide anions (as are found atphysiological concentration). Accordingly, it is expected that otherenzymes having the three types of copper centers would also be useful ascomponents of cathode electrocatalysts in cathodes operating under atnear neutral pH.

The redox potentials of the redox polymers that “wire” the cathodeenzyme can be tailored for use in the invention. Redox polymers for usein the method may include PAA-PVI-[Os(4,4′-dichloro-2,2′-bipyridine)₂Cl]^(+/2+) which can be prepared asfollows: 4,4′-Dinitro-2,2′-bipyridine N,N′-dioxide was prepared asdescribed in Anderson, S.; Constable, E. C.; Seddon, K. R.; Turp, E. T.;Baggott, J. E.; Pilling, J. J. Chem. Soc., Dalton Trans. 1985,2247-2250, and Kenausis, G.; Taylor, C.; Rajagopalan, R.; Heller, A. J.Chem. Soc., Faraday Trans. 1996, 92, 4131-4135.4,4′-dichloro-2,2′-bipyridine (dcl-bpy) was synthesized from4,4′-dinitro-2,2′-bipyridine N,N′-dioxide by modifying the procedure ofMaerker et al. (see Anderson, S., supra and Maerker, G.; Case, F. H. J.Am. Chem. Soc. 1958, 80, 2475-2477). Os(dcl-bpy)₂Cl₂ was prepared asfollows: (NH₄)₂OsCl₆ and “dcl-bpy were dissolved in ethylene glycol in a1:2 molar ratio and refluxed under argon for 1 hour (yield 85%). TheOs(dcl-bpy)₂Cl₂ was then complexed with the 1:7polyacrylamide-poly(N-vinylimidazole) (PAA-PVI) copolymer and purifiedas described in Zakeeruddin, S. M.; D. M. Fraser, D. M.; Nazeeruddin,M.-K.; Gratzel, M. J. Electroanal. Chem. 1992, 337, 253-256 to form thePAA-PVI-Ps(4,4′-dichloro-2,2′-bipyridine)₂Clr^(+/2+) redox polymer.Those skilled in the art are aware of numerous variations that can beprepared and used as redox polymers according to the invention.

Determination of the HbA1c Percentage

Once the total hemoglobin has been measured, the HbA1c/Hb ratio can bedetermined by separating the HbA1c fraction from the sample. The HbA1c,which can be converted to HbAlcO2, can then be measured indirectly andelectrochemically using the same method as for the total hemoglobin.Alternatively, these fructosyl amines may be subject to direct enzymecatalyzed electro-oxidation, for example using fructosyl amine oxidaseshaving FAD/FADH reaction centers, or by one of the copper enzymes.

The following are examples of suitable methods which incorporate theseparation and HbA1c assay steps.

Example 1

Affinity gel columns can be used to separate bound, glycosylatedhemoglobin from the nonglycosylated fraction. The gel containsimmobilized m-aminophenylboronic acid on cross-linked, beaded agarose.The boronic acid first reacts with the cis-diol groups of glucose boundto hemoglobin to form a reversible 5-membered ring complex, thusselectively holding the glycosylated hemoglobin on the column. Next, thenonglycosylated hemoglobin is eluted. The ring complex is thendissociated by sorbitol, which permits elution of the glycosylatedhemoglobin. Using affinity chromatography, absorbances of the bound andnonbound fractions, measured at 415 nm, are used to calculate thepercent of glycosylated hemoglobin.

Example 2

Magnetic beads that are <1 μm (available from Bangs Laboratories), onwhich antibodies against HbA1c would be immobilized, can be mixed with acitrate-solution diluted blood sample. Two measurements are performed,one on the entire sample, and a second on the re-oxygenated Hb1Ac boundto the magnetic beads, after their removal to a chamber of anelectrochemical cell. Alternatively, the second measurement can be onthe residual Hb, after the magnetic separation of the bead-bound HbA1c.

Example 3

Two samples of the lysed red blood cells in citrate buffer can becoulometrically assayed in two chambers. In Chamber 1, the total HbO₂would be measured. Chamber 2 contains the immobilized HbA1c-specificantibody. Either of the two would capture HbA1c without capturing Hb.After rinsing or passage of citrate buffer through Chamber 2 (e.g. byrepeated filling through capillary action and touching the edge of thechamber to filter paper), the chamber would contain only HbA1cO₂. TheHbA1cO₂ would be assayed electrochemically (preferably coulometrically)by its electroreduction, 4H⁺+4e⁻+HbA1cO₂→2H₂O+HbA1c. The HbA1c/Hb ratiocan then be calculated from the two coulometric measurements.

Example 4

As in example 3 above, except that the two coulometric measurementswould be performed in a single chamber. The chamber, which would containthe immobilized HbA1c capture agent, would be filled with a citratesolution of the lysed red blood cells. First, the total HbO₂ would beelectrochemically (preferably coulometrically) measured. Next, theunbound Hb, but not the bound HbA1c, would be rinsed out, the HbA1cwould be re-equilibrated with air, and its amount would becoulometrically measured.

Thus, the assay of the invention, in one form, can comprise a method ofdetermining the ratio of HbA1c to total Hb in blood, the methodcomprising obtaining a blood sample; electrochemically determining thetotal amount of hemoglobin in the sample, or in a treated portion of thesample; electrochemically determining the amount of HbA1c in the sample;and calculating the ratio of HbA1c to total hemoglobin. In a preferredform the method of electrochemically determining the total amount ofhemoglobin in the sample is accomplished by placing the sample in anelectrochemical cell in which, at the cathode, a cathode enzyme isbound, for example using a redox polymer. In this method, it ispreferred that the enzyme be a laccase or a bilirubin oxidase which willelectroreduce oxygen bound to the hemoglobin to water. The hemoglobincontent is determined from the oxygen content.

In another form of the invention the electrochemical determination ofHbA1c fraction can be accomplished by one of two methods. In a firstmethod, the A1c containing fraction of the hemoglobin is separated byphysical means, such as by use of an HbA1c specific antibody. Underappropriate conditions the HbA1c then present in the form of HbA1cO₂ canthen be electrochemically determined by electroreduction of the oxygen.(again with an enzyme selected to accomplish the four electron reductionof oxygen). In a second method, the glycated protein (a fructosyl amine)can be directly oxidized on cross-linked poly(N-vinyl imidazole) basedredox polymer films (without an enzyme) of sufficiently positiveoxidizing potential. Alternatively, enzymatic electrooxidation of thefructosyl amines can be used for this part of the determination.

Finally, the invention comprises an electrochemical method for thedetermination of HbA1c (or HbA1c/Hb ratio) comprising determining from astarting sample, in an electrochemical cell, the total amount ofhemoglobin (e.g. by measuring bound oxygen), separating the HbA1ccomponent from the sample using an HbA1c capturing agent, and measuringhemoglobin content in the captured or non-captured portion of thesample.

Devices for accomplishing the method of the invention are preferablysmall. By incorporating electrochemical steps, it may be possible toprepare biosensor strips which include a cathode at which the chemistrydiscussed herein is placed, as well at which the necessary anode isconstructed. Such strips can be prepared using techniques presently usedfor making commercially available biosensor strips that are used forglucose determinations, such as the FreeStyle blood glucose system soldby TheraSense, Inc. Samples could then be applied to these strips andthe strips placed in the measuring instrument (meter) to be “read.” Byconstructing a portion of the equipment in the form of electrochemicalbiosensor strips, the electrochemical method of the invention provides asignificant potential advantage of creating a smaller analysis devicewhile providing accurate results.

What is claimed is:
 1. A device for use in assaying glycated hemoglobinin a blood, blood-containing, or blood-derived sample, comprising: anelectrochemical cell comprising: a cathode comprising an enzyme capableof reducing oxygen to water; an anode; and a glycated hemoglobinhydrolysis agent or a glycated hemoglobin separating agent.
 2. Thedevice of claim 1, further comprising a spectrophotometer fordetermining an amount of glycated hemoglobin in the sample, a glycatedhemoglobin hydrolysis agent, or a glycated hemoglobin separating agent.3. The device of claim 1, further comprising an enzyme that oxidizesfructosyl amines or a cross-linked poly N-vinyl imidazole based redoxpolymer film.
 4. The device of claim 1, wherein the electrochemical cellcomprises a single chamber.
 5. The device of claim 1, wherein thecathode further comprises a redox mediator.
 6. The device of claim 5,wherein the cathode enzyme is a four copper-ion containing copperenzyme.
 7. The device of claim 6, wherein the four copper-ion containingcopper enzyme is selected from a laccase, a bilirubin oxidase,ceruloplasmin and ascorbate oxidase.
 8. The device of claim 7, whereinthe enzyme is a bilirubin oxidase.
 9. The device of claim 8 wherein theredox mediator ispolyacrylamide-poly(n-vinylimidazone)-[Os(4,4′-dichloro-2,2′-bipyridine)₂Cl]^(+/2)(PAA-PVI-[Os(4,4′-dichloro-2,2′-bipyridine)₂Cl]^(+/2+)).
 10. The deviceof claim 1, wherein the glycated hemoglobin hydrolysis agent is aproteolytic enzyme.
 11. The device of claim 10, comprising an enzymethat oxidizes ketoamines.
 12. The device of claim 11, wherein the enzymethat oxidizes ketoamines is selected from a fructosamine oxidase and afour copper-ion containing copper enzyme.
 13. The device of claim 12,further comprising a redox mediator.
 14. The device of claim 13, whereinthe redox mediator is an Os^(2+/3+) or Ru²⁺³⁺ complex.
 15. The device ofclaim 1, wherein the separating agent is selected from an anti-glycatedhemoglobin antibody and a boronic acid-modified surface.
 16. The deviceof claim 15, wherein the separating agent is an immobilizedanti-glycated hemoglobin antibody.
 17. The device of claim 16, whereinthe boronic acid modified surface is selected from magnetic, polymer orglass beads.
 18. The device of claim 1, further comprising an enzymethat oxidizes fructosyl amines.
 19. The device of claim 18, wherein theenzyme is selected from a fructosyl amine oxidase or a four copper-ioncontaining copper enzyme.
 20. The device of claim 1, further comprisinga cross-linked poly N-vinyl imidazole based redox polymer film.
 21. Thedevice of claim 1, wherein the cell comprises both a glycated hemoglobinhydrolysis agent and a glycated hemoglobin separating agent.
 22. Thedevice of claim 21, wherein the glycated hemoglobin hydrolysis agent isa proteolytic enzyme.
 23. The device of claim 22, further comprising anenzyme that oxidizes ketoamines.
 24. The device of claim 23, wherein theenzyme that oxidizes ketoamines is selected from a fructosamine oxidaseand a four copper ion containing copper enzyme.